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. 2015 Apr 29;2:15014. doi: 10.1038/mtm.2015.14

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Copyright © 2015 American Society of Gene & Cell Therapy

This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/4.0/

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LPA also enhances nuclear levels of YAP and promotes proliferation in B4G12 cells. (a) The dose-dependent stimulation of nuclear YAP in postconfluent day 4 B4G12 cells by LPA was measured by western blotting. B4G12 cells were serum starved for 24 hours and incubated in the presence of LPA at different doses for an additional 4 hours. Nuclear proteins (Nu) were extracted from each experimental group and fractionated in 10% SDS–PAGE gels (5 μg/lane). After being transferred to membranes, the proteins were subjected to blotting with an antibody against either YAP or histone H3, as a loading control. (b) The distribution of nuclear YAP (green) was detected via an immunofluorescein assay in postconfluent day 4 B4G12 cells 4 hours after 20 μmol/l LPA was added. (c) B4G12 cells were serum starved for 24 hours and treated with LPA (20 μmol/l). After treatment, from day 2 to day 6, cell growth was analyzed through cell counting (n = 3; **P < 0.01). (d) B4G12 cells treated with LPA (20 μmol/l) showed a hexagonal morphology under phase microscopy, with a normal immunostaining pattern of ATPase and ZO-1, but without any expression of SMA, indicating no evidence of endothelial–mesenchymal transition. The cell nuclei were counterstained with Hoechst 33342 (blue). LPA, lysophosphatidic acid; SMA, smooth muscle actin; YAP, yes-associated protein.